High Risk of Gestational Trophoblastic Neoplasia Development in Recurrent Hydatidiform Moles with NLRP7 Pathogenic Variations

RHM patients who were referred to the Department of Gynecologic Oncology (Tepecik Training and Research Hospital) and to the Center for Genetic Diagnosis (Dokuz Eylul University) between the years 2018 and 2020 were retrospectively reviewed. All 3 cases were included in the study. In addition to the gynecologic work up, Sanger Sequencing of the NLRP7, KHDC3L coding regions and exon-intron boundaries were performed. This study was in conformity with the Declaration of Helsinki on ethical principles for medical research. All individuals provided written informed consent for molecular analysis and also for the publication of this paper.

Total genomic DNA was extracted from 4 ml peripheral blood from all patients via magnetic bead purification method. MagPurix Blood DNA Extraction Kit (Zinexts, Taiwan) was used with the MagPurix Automated Extraction System (Zinexts, Taiwan) according to the manufacturer’s protocol. DNA quality and concentration measurements were performed by NanoDrop ND1000® Spectrophotometer (Thermo Fisher Scientific, USA). After proper quality (50-100 ng/µl concentration and A260/ A280: 1.8-2.0 purity) of DNA was ensured, the DNA was stored at -20°C until further use.

Direct amplification and sequencing of the KHDC3L and NLRP7 genes were performed primarily using the primer sequences shown on Table 1. Exons and splice-site junctions were amplified using a standard PCR procedure that utilized the AmpliTaq Gold™ 360 DNA Polymerase (Applied Biosystems - Thermo Fisher Scientific, USA). Amplifications were performed using Eppendorf 5332 Mastercycler (Eppendorf AG, Germany). PCR products were verified by 2% agarose gel electrophoresis and ethidium bromide staining. Sequencing reactions were performed with BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems - Thermo Fisher Scientific, USA), and electrophoresed on an ABI 3130 Capillary Electrophoresis System (Applied Biosystems - Thermo Fisher Scientific, USA). Sequence alignment and evaluation were performed using CLC Genomics Workbench v3.6.5 (Qiagen, Germany). GRCh38.p13 human genome reference in the “Ensembl” database, with a ENST00000370367 transcript of the KHDC3L gene and a ENST00000592784 transcript of the NLRP7 gene as reference. Detected variants were classified according to the current guidelines [8].

The first case was a gravida 3, para 0 (one early pregnancy loss and two consecutive HMs) 25-year-old woman. Her main complaint was amenorrhea. β-hCG level was measured as 271,793 mIU/mL. Ultrasonography (USG) revealed a 50 mm empty gestational sac with an irregular contour and there was no yolk sac. Several hypoechoic areas in the endometrial cavity were seen. Suction evacuation was performed without complications. Pathologic investigation revealed HM. Monitoring of β-hCG levels was initiated. Eighteen days after the evacuation, β-hCG levels lowered to 365 mIU/mL, but 7 days later it rose to 586 mIU/mL. The increase of β-hCG is considered as a sign of GTN development. The diagnosis was stage 1, low risk GTN with a WHO score of 1. Single agent chemotherapy protocol consisting of four doses of methotrexate and four doses of folinic acid was started. After 3 cycles of chemotherapy, β-hCG levels returned to the normal range after 40 days, and the treatment was concluded with one additional cycle of chemotherapy.

One and a half years after the initial management of the case, the patient was admitted to our clinic again, due to the suspicion of another pregnancy. The β-hCG level was 258,164 mIU/mL. A hyperechogenic lesion (50x32 mm) containing multiple millimetric cystic areas was seen in USG evaluation. Evacuation procedure was performed without complications and the β-hCG level was normal after two months. The material derived from evacuation was reported as HM by the pathology laboratory.

Because the case had four consecutive HM without any live births, genetic consultation was needed. Family history revealed that the patient had 9 siblings. Only one of her siblings was married and although she had 7 children, she had no history of molar pregnancy.

Sanger sequencing of the patient’s DNA identified homozygous, c.2471+1G>A (rs104895505) pathogenic variation (Guideline criteria: PVS1, PM2, PP3) [8] in 7^th intron of the NLRP7 gene (Figure 1).

Figure 1.

The second patient was a gravida 3, para 0 (three consecutive HMs) 19-year-old woman. The first admission of the patient was on suspicion of pregnancy. Her β-hCG level was 440,873 mIU/mL. During USG, there was a HM in nodular form (81x47x84 mm) which included numerous millimetric cystic degeneration areas. It was located at corpus and fundus filling the endometrial cavity, with deep myometrial infiltration close to the serosa. Thyroid function related hormones were measured out of the normal range due to an increase in the β-hCG level: free T3 was 7.19 pg/mL, free T4 was 2.17 ng/dL, TSH was 0.02 mIU/mL. Two months after the evacuation of the products of conception, her β-hCG level was 9.63 mIU/mL. The pathology report was consistent with HM.

Eight months later, she had another admission because of amenorrhea. The USG revealed a 66x52 mm lesion in the endometrial cavity, compatible with HM. The β-hCG level was 197,328 mIU/mL and thyroid function tests were also impaired: free T3 was 4.53 pg/mL, free T4 was 1.86 ng/dL and TSH was 0.02 mIU/mL. One and a half months after the evacuation procedure, her hormone profile was normal. Pathology confirmed HM.

Her third admission was made 2 years later, due to a suspected pregnancy. The β-hCG value was 272,283 mIU/ mL. Free T3 was 5.19 pg/mL, free T4 was 1.48 ng/dL and TSH was 0.02 mIU/mL. The lesion measured 66x40 mm and it was consistent with HM again. Evacuation of the products of conception was performed, and β-hCG monitoring was initiated. The β-hCG level was 2.98 at week 3 and increased to 3.66 at week 4. It increased again to 9.35 at week 5. The lesion was considered as post-molar stage 1, low risk GTN and single agent chemotherapy (methotrexate) was planned. After two cycles of chemotherapy, β-hCG was normal and two more cycles of chemotherapy were given. After treatment, free T3 was measured as 3.97 pg/mL, free T4 was 0.83 ng/dL and TSH was 0.37 mIU/ mL.

The patient had homozygous c.2471+1G>A (rs104895505) pathogenic variation in the NLRP7 gene. This variation was identical to the variation found in case 1. Although the patients were specifically questioned, no blood relation was found between them.

The third patient was a gravida 2, para 0 (two consecutive HMs) 33-year-old woman. She was admitted to the hospital because of a suspected pregnancy. Her β-hCG level was 573,347 mIU/mL. USG revealed 70x80 mm HM with cystic areas and without any fetus. One week after the evacuation procedure the β-hCG level measured 11,758 mIU/mL. The patient chose not to be followed-up after the procedure. The pathology report confirmed HM. One and half months later, the patient was readmitted to another hospital where curettage was performed. Monitored β-hCG showed a decrease to 137 mIU/mL, followed by a plateau and consequent increase to 166 mIU/mL. The case was considered a Stage 1 Low Risk GTN, and single agent chemotherapy was started. A total of two cycles of methotrexate were given. After one year of a contraception period, the patient decided to try another pregnancy and folic acid prophylaxis was given. Following conception, the β-hCG level was 276,400 mIU/mL and USG revealed HM with cystic areas and a diameter of 50 mm. Following evacuation, the β-hCG values were normal after 1.5 months. Pathology confirmed HM. Genetic evaluation by Sanger sequencing identified a homozygous c.2571dupC, p.Ile858HisfsTer11 (rs766731093) pathogenic (Guideline criteria: PVS1, PM1, PM2) frameshift variant in 8^th exon of the NLRP7 gene (Figure 2).

Figure 2.